Journal Article

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Buitrago, Lorena, Negri, Ana, Consortium, ThromboGenomics, Filizola, Marta, Simeoni, Ilenia, Rendon, Augusto, Liang, Yupu, Ouwehand, Willem H., Coller, Barry S.

2015

Overview

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants. Significance Next-generation sequencing is identifying millions of novel gene variants, presenting challenges to researchers and clinicians. Variations in the genes ITGA2B and ITGB3 affect integrin αIIbβ3, leading to the bleeding disorder Glanzmann thrombasthenia. We analyzed novel missense variants on ∼32,000 alleles of ITGA2B and ITGB3 and found missense variants affecting ∼10% of the amino acids in each protein in ∼1.3% of the population. Almost all variants are rare, indicating recent entry into the population. Two novel variants we predicted would be deleterious profoundly affected recombinant protein expression. At cut-off values that correctly predicted at least 69% of the known Glanzmann thrombasthenia mutations as deleterious, three variant prediction algorithms predicted that at least 27% of the novel variants are deleterious.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: algorithms / Alleles / amino acids / Biological Sciences / Databases, Nucleic Acid / Exome - genetics / Fibrinogen - chemistry / Fibrinogen - metabolism / Gene Frequency / HEK293 Cells / hemorrhage / high-throughput nucleotide sequencing / High-Throughput Nucleotide Sequencing - methods / Humans / Immunoblotting / integrins / Models, Molecular / mutation / Mutation, Missense / Platelet Glycoprotein GPIIb-IIIa Complex - chemistry / Platelet Glycoprotein GPIIb-IIIa Complex - genetics / Platelet Glycoprotein GPIIb-IIIa Complex - metabolism / PNAS Plus / prediction / Protein Binding / Protein Conformation / Protein Structure, Tertiary / protein synthesis / recombinant proteins / researchers / Thrombasthenia - genetics / Thrombasthenia - metabolism